Choline Kinase (ChoK) is a phosphotransferase which acts by phosphorylating choline to phosphocholine (PCho) as the first enzyme of the phosphatidylcholine (PC) synthetic pathway (also known as the Kennedy pathway). Adenosine-triphosphate (ATP) is the phosphate group donor.
As early as the 1980s empirical evidence began emerging implicating choline kinase in tumor development and progression. Phosphocholine, which is produced by choline kinase, was discovered to be a signal unique to lung tumors. In 2002, researchers examined 43 lung tumor tissues and adjacent normal lung tissues and appeared to confirm that choline kinase expression was increased in some neoplastic lung tissues. A global analysis of 167 non small cell lung cancer patients' tumors then appeared to establish an association between choline kinase over-expression and poor clinical outcome. In addition to lung cancer, various researchers have confirmed choline kinase over-expression and increased activity in tumors of the colon, breast, prostate and ovaries. Choline kinase expression and activity also have been reported to be associated with poor prognosis in other cancer types such as breast cancer. Choline-based radiopharmaceuticals, including 11C-choline and 18F-choline, are actively being studied in clinical trials for diagnostic utility in cancer patients using positron emission tomography. Based on these studies, choline kinase has been proposed as a prognostic marker for cancer progression and a potential target for the development of novel cancer chemotherapeutic agents. Other research has pointed to a role for activated choline kinase as a metabolic requirement for neoplastic growth and survival. Insulin, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, prolactin, estrogens and hypoxia-inducible factor-1α appear to be needed for the survival, growth and invasiveness of human cancers, and have all been typically found to stimulate choline kinase activity and increase intracellular phosphocholine. Growth factors can engage receptor-tyrosine kinases which stimulate two key signal transducers, the small GTPase Ras and the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). These signal transducers then can stimulate an intersecting network that activates untethered cell growth, survival and invasiveness without influence from environmental cues and, when mutated, initiate tumors in humans.
In addition, oncogenic transformation mediated by Ras oncogenes induces high choline kinase activity levels resulting in an abnormal increase in the intracellular levels of its product, PCho. Ras gene proto-oncogenes encode a protein family of small membrane-bound GTPases which appear to be involved in cellular signal transduction from outside the cell to inside the nucleus. Activation of Ras signaling causes cell growth, division, terminal differentiation and senescence. Mutations in Ras are heavily implicated in the development of cancers. It is hypothesized that mutations may permanently activate Ras. Ras oncogenic transforming potential is acquired with point amino acid substitution mutations in codons 12, 13 or 61. These Ras mutations are found in up to approximately 6.5% of breast cancers, 30% of non-small cell lung cancers, 50% of colon cancers, and 100% of pancreatic cancers. Even in the absence of these mutations the Ras signaling pathway may be central to cancer development and progression, since several Ras pathway proteins upstream (e.g. epidermal growth factor receptor and Her2/neu) and downstream (e.g. Akt, ERK kinase) of Ras are also found to be amplified or mutated in human tumors. For example, although Ras is rarely found in mutated form in breast tumors, Ras overexpression and amplification has been observed in 50-70% of breast adenocarcinomas.
Complementary findings also support the role of ChoK in the generation of human tumors. For example, nuclear magnetic resonance (NMR) techniques have shown the presence of high PCho levels in several human tumor tissues including breast, prostate, brain and ovarian tumors with respect to normal tissues. ChoK appears to be activated by multiple growth factors and signal transducers that may be regulators of neoplastic growth and survival and may be implicated in the initiation and progression of human cancers.
Evidence for choline kinase activity in cancer has also been obtained from the observation that siRNA silencing of choline kinase mRNA expression by MDA-MB-231 breast adenocarcinoma cells reduces intracellular phosphocholine, which in turn decreases cellular proliferation and promotes differentiation. Although these studies were not conducted in vivo, they nevertheless supported the validity of choline kinase as a molecular target for the development of anti-breast cancer agents.
Ras is one of the most intensely studied oncogenes in human carcinogenesis and ChoK inhibition has been hypothesized as an anti-tumor strategy with some success. The design of compounds directly affecting ChoK activity or the enzyme activated by phosphorylcholine has provided agents with anti-tumor effects in cells transformed by oncogenes, however the specific test drugs available to-date suffer from delivery and/or safety deficiencies which make them unsuitable for clinical use.
Several ChoK inhibitors are well-known in the art. Researchers identified Hemicholinium-3 (HC-3) as a relatively potent and selective blocking agent (Cuadrado A., et al., 1993, Oncogene 8: 2959-2968, e.g.). HC-3 is a choline homologue with a biphenyl structure and has been used for designing new anti-tumor drugs. However, HC-3 is a potent respiratory paralyzing agent and is therefore not a good candidate for its use in clinical practice. Introduction of structural modifications have reduced toxic side effects but full retention of inhibitory activity is not achieved. Bisquaternized symmetric compounds derived from pyridinium have also been found to inhibit PCho production in whole cells (WO98/05644). However, these derivatives have high toxicity levels limiting extended therapeutic application. ChoK-specific siRNAs have been developed but use of an siRNA is not feasible due to a lack of suitable technology for transporting the siRNA to the tumor cell, and due to lack of selectivity of inhibition among ChoK isoforms. Mori et al (Cancer Res., 2007, 67:11284-11290).
Hence, there remains a need in the art for pharmaceutical compounds which effectively inhibit ChoK-alpha while reducing the toxic side effects which accompany the current state-of-the-art.